Inulin is a fructan-type carbohydrate, consisting mostly of fructose units, which occurs in many plants as a reserve carbohydrate. Inulin can be produced by certain bacteria and can also be enzymatically produced in vitro from sucrose. Inulin naturally occurs as a polydisperse mixture of carbohydrate molecules which are essentially composed of fructosyl units forming chains in which the fructosyl units are mainly or exclusively linked to one another by a ß(2,1) bound. The mainly linear chains are possibly bearing one or more side chains essentially composed of fructosyl units, thus forming branched inulin molecules with a fructosyl-fructosyl linkage at the branching point commonly formed by a fructosyl-fructosyl ß(2,6) bound. Inulin molecules from plant origin mostly contain one terminal glucosyl unit. Accordingly, inulin molecules can be represented by the formula GFn or Fm wherein G represents a terminal glucosyl unit, F represents a fructosyl unit and n and m represent the number of fructosyl units linked to one another through a ß(2,1) and/or a ß(2,6) bound. The number n+1, respectively m, indicates the degree of polymerisation (DP) of the inulin molecule. Inulin is further characterised by its (number) average degree of polymerisation, represented by (DP). This is the value which corresponds to the total number of saccharide units (G and F units) in a given inulin sample divided by the total number of inulin molecules in said sample, without taking into account the monosaccharides glucose (G) and fructose (F) and the disaccharide sucrose (GF) which are possibly present in the sample. The average degree of polymerisation (DP) is commonly determined by the method described by De Leenheer et al. (1).
Native inulin from plant sources (i.e. the inulin as present in the plant) appears as a polydisperse mixture of mainly linear polysaccharide chains with a (DP) ranging from 2 to about 100, whereas inulin molecules from bacterial origin, which commonly are branched ones, usually have much higher (DP) values, even up to about 115.000. Plant inulin has a (DP) which largely depends on the plant source and on the harvest, storage and processing conditions. Natural (or standard grade) inulin indicates herein inulin which has been extracted from plant sources, purified and isolated, without applying a treatment for reducing or increasing its (DP) and it usually has a (DP) which is about 1 unit lower than the (DP) of the corresponding native inulin.
Inulin molecules with a low degree of polymerisation, usually defined as a (DP)<10, are named inulo-oligosaccharide(s), fructo-oligosaccharide(s) or oligofructose. These terms, including linear and branched inulin of (DP)<10, are commonly, also herein, used interchangeably. Oligofructose is also termed herein short-chain inulin.
Inulin is commonly manufactured from plant sources, mainly from roots of Chicory (Cichorium intybus), but also from tubers of Jerusalem artichoke (Helianthus tuberosus) and from the piña (head) of the Blue Agave plant, in which inulin can be present in concentrations up to about 20 wt % on fresh plant material (hereinafter wt % means percent by weight). Inulin can be readily extracted from said plant parts and purified according to conventional techniques.
Natural inulin from chicory, respectively from J. artichoke, commonly appears as a polydisperse mixture of slightly branched chains (typically chains with less than 2 percent, respectively less than 1 percent, branching) with a (DP) ranging from 2 to about 70, respectively from 2 to about 40. Natural (standard grade) chicory inulin has a (DP) of about 10 and natural (standard grade) inulin from J. artichoke has a (DP) of about 6.
Natural inulin from agave appears as a polydisperse mixture of highly branched chains with a (DP) commonly ranging from about 14 to about 17.
At industrial scale, chicory inulin is conventionally obtained by extraction of shredded chicory roots with hot water yielding a crude inulin solution which is subsequently purified by depuration (treatment with lime followed by carbonatation and filtration) and by refining (involving treatment over ion-exchangers, treatment with active carbon and filtration). Standard grade inulin is then commonly obtained from the purified and refined solution by spray-drying. Optionally, monomeric and dimeric saccharides are removed from the purified and refined solution (e.g. by column chromatographic separation as described in EP 0670 850) to yield via spray-drying an inulin grade with a standard (DP) of about 10 which is about free of monomeric and dimeric saccharides. Optionally the purified and refined solution can be fractionated to remove monomeric and dimeric saccharides as well as oligofructose (e.g. by directed crystallisation as described in EP 0 769 026) and the fractionated inulin is then isolated in particulate form by spray-drying. Depending on the manufacturing process, chicory inulin with a (DP) ranging from about 10 (standard grade) to about 30, and even more, can be obtained.
Similarly, agave inulin can be obtained at industrial scale by squeezing, or extracting with water, shredded heads or pulp from Blue Agave, followed by conventional purification, refining and isolation of the inulin e.g. via spray-drying.
Inulin, including linear and branched inulin, with a (DP)≥20 is termed herein long-chain inulin, whereas linear and branched inulin with a (DP) from 10 to <20 is termed herein medium-chain inulin.
Inulin from chicory is for example commercially available as RAFTILINE® from ORAFTI (Tienen, Belgium) in various grades. Typical grades are RAFTILINE® ST (with a (DP) of about 10 and containing in total about 8% by weight glucose, fructose and sucrose) and RAFTILINE® HP (with a (DP) of at least 20, commonly with a (DP) of about 23 to about 25, and virtually free of glucose, fructose and sucrose).
Agave inulin is commercially available, for example industrial grade agave inulin as GAVEDIET® PR with a (DP) of 14-16 and containing in total about 5% by weight of glucose and fructose, from Industrias Colibri Azul S. A. de C.V., Mexico.
Oligofructose can be obtained according to techniques which are known in the art, including enzymatic in vitro synthesis from sucrose, as for example described in U.S. Pat. No. 5,314,810, and partial hydrolysis of inulin, as for example described in EP 0 917 588.
Oligofructose prepared by enzymatic hydrolysis of chicory inulin is commercially available in various grades, for example as RAFTILOSE® from ORAFTI (Tienen, Belgium), e.g. RAFTILOSE® L95 (liquid form) or RAFTILOSE® P95 (powder form), both with a content of about 95% oligofructose (% is wt % on total carbohydrates) with a (DP) from 2 to 9, typically with a (DP) mainly from 2 to 7, a (DP) of about 4.5, and containing about 5% in total (% is wt % on total carbohydrates) of glucose, fructose and sucrose, and RAFTILOSE® L85, liquid form with a content of about 85% oligofructose (% is wt % on total carbohydrates) with a (DP) from 2 to 9, typically a (DP) mainly from 2 to 7, a (DP) of about 3.5, and containing about 15%, maximally 20% in total (% is wt % on total carbohydrates) of glucose, fructose and sucrose.
Unless otherwise specified, the term inulin used herein refers to linear as well as branched inulin, and includes inulin molecules with a (DP)<20 as well as inulin molecules with a (DP)≥20.
In the food and feed industry, oligofructose is widely used as a low-calorie partial or complete replacement for sugar, providing sweetness, body and mouthfeel, whereas inulin of a (DP) of at least about 10, preferably of at least 20, is utilised (i) as a partial or complete low-calorie replacement for sugar in combination or not with one or more high intensity sweeteners, providing body and mouthfeel, (ii) as a texture improver, and (iii) as a low-calorie replacement for fat. The use of inulin as fat replacer results from the fact that inulin can form with water a particle gel with a stable, homogeneous, creamy structure with excellent organoleptic properties.
Inulin molecules with a (DP)≥10 as well as oligofructose molecules with a (DP)<10, are not hydrolysed by human digestive enzymes. Accordingly, these molecules pass the upper part of the digestive tract and the small intestine unaltered (Ellegård et al. (2)) and reach almost quantitatively the large intestine where they are fermented by specific intestinal bacteria (Roberfroid et al. (3)). As a result thereof, inulin and oligofructose present highly interesting nutritional properties.
Firstly, inulin and oligofructose are considered as dietary fibres. They reach the large intestine unaltered, thus providing carbon energy to the microflora in the large intestine. In this manner, inulin and oligofructose are stimulating the growth of gut bacteria in the large intestine which has a beneficial effect on the gut function, including a bulking effect (i.e. increase of the bacterial biomass) which in turn results in an increased stool weight, an increased stool frequency and a relief of constipation (Roberfroid (4)).
Furthermore, it has been found that inulin and oligofructose have a strong bifidogenic effect because inulin and oligofructose selectively stimulate the growth and metabolic activity of Bifidobacteria and Lactobacilli. Besides, while the counts of intestinal Bifidobacteria are significantly increased by the oral intake of inulin or oligofructose, a concomitant significant reduction of the counts of undesirable or pathogenic bacteria, such as e.g. Clostridia and Escherichia, in the large intestine has been observed (Gibson et al. (5) and Wang (6)). The intake of inulin and oligofructose thus largely modifies and modulates the gut flora by selectively increasing colonisation of the large intestine by beneficial bacterial species, typically Bifidobacteria, while suppressing the growth of undesirable bacterial species, which in turn results in favourable prophylactic and therapeutic effects on intestinal disorders of the host.
In vivo experiments with healthy volunteers showed inulin (RAFTILINE® ST) and oligofructose (RAFTILOSE® P95) to be bifidogenic to the same extent (Gibson et al. (7)), while in vitro experiments revealed that inulin ((DP)≥10) is fermented in the large intestine about twice as slowly as oligofructose ((DP)<10) (Roberfroid et al. (3)).
From these observations it follows that oligofructose is almost completely fermented in the proximal part of the large intestine, i.e. the ascendent part, whereas inulin is likely to reach to a more or lesser extent also more distal parts of the large intestine, i.e. the transversal and descendent parts, where it is fermented.
In vitro tests revealed that agave inulin is about as easily fermented as oligofructose. Accordingly, it is assumed that agave inulin is also almost completely fermented in the proximal part of the large intestine of humans and mammals.
Moreover, it has been disclosed that oligofructose and inulin have preventive and therapeutic effects with respect to the genesis and growth of certain cancers such as colon cancer (WO 98/52578) and mammary cancer (EP 0 692 252).
The effects against mammary cancer seem to be related to an immuno-modulating effect, particularly a stimulating effect on the immune system, of oligofructose, inulin and/or their fermentation products, mainly short chain fatty acids (SCFA) (Namioka et al. (8)).
With respect to colon cancer (usually resulting from pre-neoplastic lesion formation in the distal part of the colon), it has been reported that long-chain inulin, i.e. inulin with a (DP)≥20, is more effective in preventing the genesis of colon cancer and in inhibiting the growth of colon cancer, than oligofructose (with a (DP)<10) and standard grade chicory inulin (with a (DP) of about 10) (WO 98/52578).
Furthermore, it has been found in studies with healthy human volunteers who were slightly hyperlipidemic, that the consumption of oligofructose or inulin has beneficial effects on lipid metabolism since the consumption resulted in reducing the level of serum triglycerides and cholesterol (mainly LDL cholesterol) compared to a control placebo treatment (Brighenti et al. (9) and Jackson et al. (10)). Moreover, it has been demonstrated in rat experiments that the addition of oligofructose or inulin to a fat-rich diet reduced serum cholesterol as well as serum triglycerides by more than 50% compared to a control group (Kok et al. (11)).
Furthermore, positive effects of the consumption of oligofructose and inulin on the intestinal absorption of minerals, particularly calcium (Ca), magnesium (Mg) and iron (Fe), as well as on the bone mineral density (BMD), have been found in various studies.
Shimura et al. (12), Levrat et al. (13), Rémésy et al. (14), Tagushi et al. (15) and Scholz-Ahrens et al. (16) reported studies with rats in which an increased absorption of calcium, and in some cases of other minerals, including magnesium, was demonstrated as a result of oral consumption of inulin or oligofructose. Ohta et al. (17) and Baba et al. (18) formulated the hypothesis that the positive effects of non-digestible carbohydrates on Ca and Mg absorption occur at the level of the large intestine. Up to then, it was generally accepted that mineral absorption occurred mainly via the small intestine. Delzenne et al. (19) reported that a diet supplemented with 10 wt % of either inulin (RAFTILINE® ST) or oligofructose (RAFTILOSE® P95) resulted in a strong absorption increase for magnesium and calcium and a moderate absorption increase for iron in healthy rats, and noted almost the same effect for inulin (RAFTILINE® ST) compared to oligofructose (RAFTILOSE® P95). Brommage et al. (20) disclosed a similar increase in Ca absorption in healthy rats fed a diet supplemented with 5 wt % oligofructose (RAFTILOSE® P95). Taguchi et al. (15) reported that in ovariectomised rats oligofructose (2.5 wt % and 5 wt % in the diet) increased mineral uptake, particularly Ca and Mg absorption, and increased bone density thus preventing bone loss caused by oestrogen deficiency. Using the same model, Scholz-Ahrens et al. (16) observed a dose-dependent effect of oligofructose (RAFTILOSE® P95) (at 2.5; 5 and 10 wt % in the diet) on calcium absorption and on bone mineralisation. In that study, oligofructose also significantly reduced the osteoporotic loss of the bone trabecular structure caused by ovariectomy. Furthermore, an increased Ca absorption with a concurring increased BMD in rats fed a diet containing 5 wt %, respectively 10 wt %, inulin (RAFTILINE® HP) was reported by Lemort et al. (21).
The findings that inulin and oligofructose can positively influence the absorption of minerals from the diet and affect the uptake of minerals in the bone tissue, leading to increased BMD, are of high importance for human health. Indeed, calcium uptake in the body, bone mineral density increase, as well as the possibility to prevent, to slow down or to curb bone mineral density reduction, are very important for human populations with a typical Western-type lifestyle and food pattern, since in these populations there occurs with increasing age, particularly in post-menopausal women, a dysbalance between mineral uptake and mineral resorption and excretion. Said dysbalance results in a reduction of BMD and in bone fragilisation, which in a pronounced stage is known as osteoporosis. In an advanced stage, osteoporosis leads in turn to a high incidence of bone fractures. Accordingly, it is very important to ensure the building up during the growth phase of children and adolescents of skeletal elements with a high BMD. Such skeletal elements will indeed resist longer to demineralisation caused by any factor, and this may thus postpone or even avoid bone fracture due to advanced osteoporosis. In view of the above, it is also most important to be able to reduce possible losses of bone mineral content in adults in order to prevent or to maximally delay undesirable osteoporosis-related conditions, and in particular to slow down the post-menopausal demineralisation leading to osteoporosis and eventually to bone fracture. Furthermore, it is very important to be able to remedy conditions of osteoporosis, in particular in case of the occurrence of osteoporosis-related bone fractures. At last, it is highly desirable to be able to stimulate and increase mineral uptake and formation of bone structure in case of necessity, for example in case of accidental bone fractures in children, adults and elderly people.
In view thereof, the disclosures regarding the increased mineral absorption in rats have received much attention from the medical world and several studies have been made in order to examine Ca absorption from the diet and to increase Ca uptake in the bone tissue, in order to increase or improve BMD and bone structure in humans. Ellegård et al. (2) determined the mineral balance in ileostomy volunteers who were administered 15 g/day of either inulin (RAFTILINE® ST) or oligofructose (RAFTILOSE® P95). The intake of neither inulin nor oligofructose was found to alter the mineral excretion from the small intestine, thus confirming that the effect of inulin and oligofructose on mineral absorption does not occur in the small intestine but essentially takes place in the large intestine (also termed the colon). Studies by Coudray et al. (22) with healthy male adults (metabolic balance method) showed a significantly increased Ca absorption with a dietary intake of 40 g inulin per day. In studies (dual stable isotopes method) with healthy male adolescents, Van den Heuvel et al. (23) found a significant increase in Ca uptake upon consumption of 15 g/day oligofructose (RAFTILOSE® P95).
The beneficially nutritional effects resulting from the intake of oligofructose and inulin apparently are the result of their fermentation in the large intestine. However, as reported by Roberfroid et al. (3), the fermentation rate of inulin is much slower than the one of oligofructose.
Furthermore, in vitro experiments (unpublished results) with human faecal slurries even indicated to the inventors that when long-chain inulin (i.e. inulin with (DP)≥20), was essentially free from oligofructose, i.e. inulin of (DP)<10), its fermentation hardly started.
The above observations, on the one hand the improved nutritional effects of inulin, particularly of long-chain inulin, and, on the other hand, the difficult and slow start of the fermentation and the resulting low fermentation rate of inulin, particularly long-chain inulin, in the large intestine, clearly lead to a technical problem which limits and even prevents the use of long-chain inulin to maximally generate nutritional benefits in humans and mammals.
Furthermore, in most of the nutritional studies disclosed so far, a daily consumption of relatively high amounts of oligofructose or inulin have been used, namely 15 g to 40 g/day in human studies and 2.5 wt % to 10 wt % and even 20 wt % of the diet in rat studies. Extrapolated to humans, a rat diet containing 2.5 wt % to 10 wt % oligofructose or inulin would correspond to an amount oligofructose or inulin of about 15 g to 60 g/day. Such relatively high daily amounts also constitute a further technical problem for the use of inulin for nutritional purposes, particularly for generating improved beneficially nutritional effects in humans, because, as is known, such relatively high doses may cause intestinal side effects, such as too much flatulence, too much intestinal pressure, intestinal cramps and even diarrhoea.